A novel Cellvibrio mixtus family 10 xylanase that is both intracellular and expressed under non-inducing conditions

Hydrolysis of the plant cell wall polysaccharides cellulose and xylan requi res the synergistic interaction of a repertoire of extracellular enzymes. R ecently, evidence has emerged that anaerobic bacteria can synthesize high l evels of periplasmic xylanases which may be involved in the hydrolysis of s mall xylooligosaccharides absorbed by the micro-organism. Cellvibrio mixtus , a saprophytic aerobic sail bacterium that is highly active against plant cell wall polysaccharides, was shown to express internal xylanase activity when cultured on media containing xylan or glucose as sole carbon source. A genomic library of C. mixtus DNA, constructed in lambda ZAPII, was screene d for xylanase activity. The nucleotide sequence of the genomic insert from a xylanase-positive clone that expressed intracellular xylanase activity i n Escherichia coli revealed an ORF of 1137 bp (xynC), encoding a polypeptid e with a deduced M-r of 43413, defined as xylanase C (XylC). Probing a gene library of Pseudomonas fluorescens subsp. cellulosa with C. mixtus xynC id entified a xynC homologue (designated xynG) encoding XylG; XylC and xynC we re 67% and 63% identical to the corresponding C. mixtus sequences, respecti vely. Both XylC and XylG exhibit extensive sequence identity with family 10 xylanases, particularly with non-modular enzymes, and gene deletion studie s on xynC supported the suggestion that they are single-domain xylanases. P urified recombinant XylC had an M-r of 41000, and displayed biochemical pro perties typical of family 10 polysaccharidases. However, unlike previously characterized xylanases, XylC was particularly sensitive to proteolytic ina ctivation by pancreatic proteinases and was thermolabile. C. mixtus was gro wn to late-exponential phase in the presence of glucose or xylan and the cy toplasmic, periplasmic and cell envelope fractions were probed with anti-Xy lC antibodies. The results showed that XylC was absent from the culture med ia but was predominantly present in the periplasm of C. mixtus cells grown on glucose, xylan, CM-cellulase or Avicel. These data suggest that C. mixtu s can express non-modular internal xylanases whose potential roles in the h ydrolysis of plant cell wall components are discussed.